detection of the analyte is possible at lower concentrations, but
the risk of side effects is higher. Raman measurements using a
FT-spectrometer with the NIR laser offer spectra of lower
intensity and resolution, but much lower sample fluorescence
which can be the only option for the analysis of many plant
sample.
19. For samples containing a chromophore it is possible to perform
resonance Raman measurements. When the wavenumber of
the laser excitation used for Raman measurements coincides
with an electronic transition of the individual molecule, the
resonance (or preresonance) Raman effect is observed. Due to
this effect, Raman spectra of the analytes can be recorded even
when these compounds are present in the sample at very low
concentrations (down to 10
À8 M).
20. The laser power varies depending on the laser type and needs
to be adjusted for each sample. The higher the laser power, the
stronger the Raman signal observed. Too high power can cause
sample heating and water evaporation thus resulting in sample
damage (Table 1).
21. The higher the integration time, the stronger the Raman signal. Integration time is a parameter set for dispersive Raman
spectrometers. It is the exposure time of the sample to laser
light as well as the time of recording signals during a singlepoint Raman measurement. Too long integration time can
cause damage to the sample.
22. Number of accumulations is the number of individual spectra
registered at the same point (number of scans per point),
averaged over scanned points. The higher the number of accumulations, the better the Raman spectrum quality (higher
signal to noise ratio).
23. Spectral resolution set for interferometric spectrometers is
usually 2 or 4 cm
À1 . In dispersive Raman spectrometers, spectral resolution depends on the chosen type of grating (e.g.,
600 and 1800 grooves per mm providing spectral resolution of
3 and 1 cm
À1
, respectively).
24. The sampling density (an increment, a step size) defines how
many points are measured in the selected sample area. The
lower the increment, the more points per line and lines per
image are measured, and the better is the spatial resolution of
the Raman image obtained. A measurement of more points
requires longer acquisition time, so the compromise between
these two criteria, the spatial resolution and the acquisition
time, is necessary. It may be useful to measure the whole area
with a lower spatial resolution (small number of points) first
and then to repeat the measurement of a selected smaller area
with a higher spatial resolution.
Light Microscopy and Raman Imaging of Carotenoids
257
the risk of side effects is higher. Raman measurements using a
FT-spectrometer with the NIR laser offer spectra of lower
intensity and resolution, but much lower sample fluorescence
which can be the only option for the analysis of many plant
sample.
19. For samples containing a chromophore it is possible to perform
resonance Raman measurements. When the wavenumber of
the laser excitation used for Raman measurements coincides
with an electronic transition of the individual molecule, the
resonance (or preresonance) Raman effect is observed. Due to
this effect, Raman spectra of the analytes can be recorded even
when these compounds are present in the sample at very low
concentrations (down to 10
À8 M).
20. The laser power varies depending on the laser type and needs
to be adjusted for each sample. The higher the laser power, the
stronger the Raman signal observed. Too high power can cause
sample heating and water evaporation thus resulting in sample
damage (Table 1).
21. The higher the integration time, the stronger the Raman signal. Integration time is a parameter set for dispersive Raman
spectrometers. It is the exposure time of the sample to laser
light as well as the time of recording signals during a singlepoint Raman measurement. Too long integration time can
cause damage to the sample.
22. Number of accumulations is the number of individual spectra
registered at the same point (number of scans per point),
averaged over scanned points. The higher the number of accumulations, the better the Raman spectrum quality (higher
signal to noise ratio).
23. Spectral resolution set for interferometric spectrometers is
usually 2 or 4 cm
À1 . In dispersive Raman spectrometers, spectral resolution depends on the chosen type of grating (e.g.,
600 and 1800 grooves per mm providing spectral resolution of
3 and 1 cm
À1
, respectively).
24. The sampling density (an increment, a step size) defines how
many points are measured in the selected sample area. The
lower the increment, the more points per line and lines per
image are measured, and the better is the spatial resolution of
the Raman image obtained. A measurement of more points
requires longer acquisition time, so the compromise between
these two criteria, the spatial resolution and the acquisition
time, is necessary. It may be useful to measure the whole area
with a lower spatial resolution (small number of points) first
and then to repeat the measurement of a selected smaller area
with a higher spatial resolution.
Light Microscopy and Raman Imaging of Carotenoids
257